KR20150043937A - A compositon for diagnosing a liver cancer in a subject, method for diagnosing a liver cancer in a subject and method for obtaining information for diagnosing a liver cancer in a subject - Google Patents

Abstract

Provided are a composition for diagnosing liver cancer of an object comprising a material which is specifically bound to TMED2, CD43, or a combination thereof, a method for diagnosing liver cancer of the object, and a method for obtaining information needed for diagnosing liver cancer of the object.

Description

Technical Field [0001] The present invention relates to a composition for the diagnosis of liver cancer of an individual, a method for diagnosing an individual's liver cancer, and a method for obtaining information necessary for diagnosis of an individual's liver cancer diagnosing a liver cancer in a subject}

A method for diagnosing liver cancer in an individual, and a method for obtaining information necessary for diagnosis of a liver cancer in an individual.

Recently, the necessity of diagnosis through biological fluids such as blood, urine, and saliva is increasing when biopsies are difficult or difficult to diagnose diseases such as cancer. However, due to the absence of highly accurate markers, there is a difficulty in diagnosing using biological fluids. Although alpha-fetoproetin (AFP) is known for liver cancer, it is not sensitive to the detection of liver cancer and increases in patients with liver cirrhosis.

In addition, diagnosis through biopsy involves the patient's pain due to invasive procedures such as incision, and has the problem that the accuracy of diagnosis is reduced if an error occurs in sampling.

TMED2 (transmembrane emp24 domain trafficking protein 2) is a protein encoded by the TMED2 gene in humans. TMED2 is known to bind to GORASP1 and GORASP2.

CD43 (cluster of differentiation 43) is also known as sialophorin, SPN or leukosialin, and is a transmembrane cell surface protein encoded by the SPN gene in humans. CD43 is a major sialoglycoprotein on the surface of human T lymphocytes, monocytes, granulocytes, and some B lymphocytes and appears to be important for immune function and is a part of the physiological ligand-receptor complex involved in T-cell activation .

However, it has not been disclosed that TMED2, and / or CD43 is associated with liver cancer.

One aspect provides a composition for the diagnosis of liver cancer, comprising a substance that specifically binds to TMED2, CD43, or a combination thereof.

Another aspect provides a method of diagnosing liver cancer in an individual.

 Another aspect provides a method for obtaining the information needed to diagnose an individual's liver cancer.

One aspect provides a composition for the diagnosis of liver cancer, comprising a substance that specifically binds to TMED2, CD43, or a combination thereof.

TMED2 (transmembrane emp24 domain trafficking protein 2) is a protein encoded by the TMED2 gene in humans. TMED2 is known to bind to GORASP1 and GORASP2. TMED2 may be encoded by the amino acid sequence of NP_006806 (SEQ ID NO: 1) and the NM_006815 nucleotide sequence (SEQ ID NO: 2).

CD43 (cluster of differentiation 43) is also known as sialophorin, SPN or leukosialin, and is a transmembrane cell surface protein encoded by the SPN gene in humans. CD43 is a major sialoglycoprotein on the surface of human T lymphocytes, monocytes, granulocytes, and some B lymphocytes and appears to be important for immune function and is a part of the physiological ligand-receptor complex involved in T-cell activation . CD43 may be encoded by the amino acid sequence of NP_001025459 (SEQ ID NO: 3) and the nucleotide sequence of NM_001030288 (SEQ ID NO: 4).

The TMED2, CD43, or a combination thereof may be present on the surface of a microvessel, for example, a microbeptide derived from a living body. The TMED2, CD43, or a combination thereof may be specifically present on the surface of the microvesicles isolated from an individual having liver cancer. The TMED2, CD43, or a combination thereof may be present at a higher level in a sample isolated from a liver cancer subject than a microvessel isolated from a control sample. The control group may be a normal individual, a disease other than liver cancer, for example, an individual having cirrhosis, or a combination thereof. Therefore, according to the composition, liver cancer can be distinguished from normal individuals or liver cirrhosis individuals.

The separation of microvessels can be accomplished by known methods. The separation may be carried out by centrifuging the sample, filtering the sample, incubating the material capable of specifically binding to the microbicule or a material capable of interfering with the lipid bilayer of the microbicide with a microbead, or And combinations of these. The incubation can be performed in vitro. Separation of the microbicule can be carried out by, for example, separation using a solid support or centrifugal force, density gradient method, ultracentrifugation, filtration, dialysis, immunoaffinity chromatography using an antibody, free flow electrophoresis, . ≪ / RTI > The substance that can specifically bind to the microbicule may be a surface protein, a lipid, or a substance capable of binding to the sugar. Wherein the surface protein is selected from the group consisting of CD63, CD83, CD9, integrin-beta 1 (ITGB1), EpCAM, caveolin, FasL, HLA-DRA, CD36, CD63, CD81, MUC1, ERBB4, GPER, ERBB2, MLANA, AMHR2, Lt; / RTI > The substance capable of specifically binding to the microbeptide may be selected from the group consisting of a substance having binding affinity to a protein, a substrate of an enzyme, a coenzyme, a substance which specifically binds to a regulator, a receptor, a lectin, a sugar, a glycoprotein, Binding protein, a protein comprising a pleckstrin homology (PH), a cholesterol-binding protein, or a combination thereof. The antigen-binding fragment comprises an antigen binding site, for example a single-domain antibody, Fab, Fab 'or scFv. The interstitial material in the lipid bilayer of the microvessel may be a material comprising a lipophilic moiety, an amphiphilic moiety, an amphoteric ion moiety, or a combination thereof. The lipophilic moiety may be, for example, a fatty acid, a sterol, or a glyceride. The amphiphilic moiety may be, for example, a phospholipid or a sphingolipid. The amphoteric ion moiety may be, for example, sulfobetaine, carboxybetaine, or phosphorylcholine. A substance capable of specifically binding to the microvesicle or a substance capable of interfering with the lipid bilayer of the microbequilla may be a solid support. The solid support may be spherical, polyhedral, plate, linear, or a combination thereof. The solid support may be polystyrene, polypropylene, magnetic particles or a combination thereof.

The sample represents a biological material derived from an individual. The biological material may be a solid tissue, such as a fresh or preserved organ or tissue sample or biopsy; Blood or blood components; Bodily fluids such as amniotic fluid, peritoneal fluid, or interstitial fluid; Cell, or a combination thereof. The sample may include a compound that is not naturally mixed with biological materials such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Biological samples may include, for example, urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, airway fluid, intestinal fluid, genitourinary fluid, Intracranial body fluids, ascites, cystic tumor body fluids, saline fluids, or a combination thereof. The sample may be one comprising a circulating tumor cell (CTC). The substance specifically binding to TMED2, CD43, or a combination thereof may be a substance specifically binding to TMED2, CD43, or a combination thereof present on the surface of the microbeptide separated from the living body.

A substance that specifically binds to TMED2, CD43, or a combination thereof may be a substance that specifically or specifically binds to TMED2, CD43, or a combination thereof, either naturally or non-naturally. The substance that binds transiently may be a substance that specifically binds to TMED2, CD43, or a combination thereof in vivo, for example, a protein. The substance that specifically binds to TMED2, CD43, or a combination thereof may be an antibody, or an antigen-binding fragment thereof, a ligand, a substrate, an inhibitor, an agonist, an antagonist, a cofactor, or a combination thereof. The antibody may be a monoclonal antibody or a polyclonal antibody. The antigen-binding fragment of the antibody may be a single-domain antibody, Fab, Fab ', scFv or a combination thereof.

A substance that specifically binds to TMED2, CD43, or a combination thereof may be one with a detectable label attached thereto. The detectable label may be an optical label, an electrical label, a radioactive label, an enzyme label, or a combination thereof. The optical label may be a substance that generates fluorescence or phosphorescence. The fluorescent material may be, for example, fluorescein, rhodamine, cyanine, metal porphyrin complex, Cy-5 and Cy-3. Examples of fluororesin dyes include 6-carboxyl fluorosine (6-FAM) 1,2, 4 ', 1,4, -tetrahydrochloride (TET) 2 and 2', 4 ' 5 ', 7', 1,4-hexachlorofluorescein (HEX) 3, 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxydodamine (JOE) Chloro-5'-fluoro-7 ', 8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein 5 and 2'-chloro-7'- -Carboxyfluorescein 6 may be included.

The material that specifically binds to TMED2, CD43, or a combination thereof may be immobilized on a glass or solid support. The solid support may be nano or microparticles. The solid support may be magnetic or non-magnetic particles. The solid support may be a bead, a sphere, a polygon, a plate, or a combination thereof. The solid support may have the form of an array in which the material is immobilized in a particular region.

The composition may further comprise reagents for use in determining the level of expression of mir-210, mir-346, or a combination thereof. The reagent may be one which binds to mir-210, mir-346, or a combination thereof, or to a nucleotide sequence complementary to mir-210, a nucleotide sequence complementary to mir-346, or a combination thereof. The reagent may be a natural, synthetic, or semi-synthetic material. The reagent may be a nucleic acid, for example, DNA, RNA, DNA-RNA hybrid, PNA, or a combination thereof.

The reagent may include mir-210 or its complementary sequence; Or a primer, probe, or antisense sequence thereof that specifically binds to mir-346 or its complementary sequence; Or a combination thereof. The primer may be one which provides a polymerization initiation point in a polymerization reaction with a polymerase. The primer may be one used in a nucleic acid amplification reaction. The term "amplification" refers to increasing the number of copies of the target sequence or its complementary sequence. The nucleic acid amplification reaction can be performed by any method known in the art. Amplification of nucleic acids involves either a method requiring multiple cycles during amplification or a method performed at a single temperature. Examples of cycling techniques include those that require thermal cycling. Methods that require thermocycling include polymerase chain reaction (PCR). PCR is known in the art. PCR typically involves denaturing double stranded DNA to single stranded DNA by thermal degradation, annealing the primer to the single stranded DNA; And synthesizing a complementary strand from the primer. An isothermal amplification method is a method in which a single temperature or a major aspect of the amplification process is performed at a single temperature. In contrast to PCR, in which the reaction product is heated to allow additional primers to bind in order to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and rescopy the template do. Isothermal methods can be distinguished by methods that rely on substitution of primers to initiate reiterative template copying and methods that rely on sequential reuse or novel synthesis of single primer molecules. Methods that rely on primer substitution include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA) and loop mediated amplification and loop mediated amplification (LAMP). Methods that rely on sequential reuse or novel synthesis of single primer molecules include those selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA). The primers may be included in one, or two or more sets according to the amplification method selected. The primer may be a PCR primer.

mir-210 represents a mature mir-210 microRNA having the nucleotide sequence of SEQ ID NO: 5. mir-346 represents a mature mir-346 microRNA having the nucleotide sequence of SEQ ID NO: 6.

The reagent may be one with a detectable label attached thereto. The detectable label is as described above. The reagent may be immobilized on a glass or solid support. The solid support is as described above.

The composition may have any phase. The composition may be a liquid, a solid, or a combination thereof.

The subject may be a mammal. The mammal may be a human, mouse, cow, pig, horse, sheep, dog, cat, or a combination thereof.

Another aspect provides an individual liver cancer diagnostic kit comprising a substance that specifically binds TMED2, CD43, or a combination thereof.

The substances specifically binding to TMED2, CD43, or a combination thereof are as described for the claimed composition. The kit may further comprise reagents for use in the diagnosis of liver cancer in an individual. The reagent may comprise a buffer, an indicator, or a combination thereof.

Another aspect relates to a method of preparing a microbeptide comprising contacting a sample separated from an individual with a substance that specifically binds TMED2, CD43, or a combination thereof to bind the microbeptide and the substance; And measuring the level of the substance in the microbicule in the sample.

The method comprises contacting a sample separated from an individual with a substance that specifically binds TMED2, CD43, or a combination thereof to bind the microbeptide to the substance.

The contacting may be performed in a liquid medium. The liquid medium may be a liquid sample itself, water, a buffer, or a combination thereof. The contacting can be performed by mixing the sample and the material. The contacting may be performed by stirring the sample and the material in a vessel. The stirring may be performed at a level that does not destroy the microbeads.

The subject may be a mammal. The mammal may be a primate. The mammal may be a human, mouse, cow, pig, horse, sheep, dog, cat, or a combination thereof.

The sample represents a biological material derived from an individual. The biological material may be a solid tissue, such as a fresh or preserved organ or tissue sample or biopsy; Blood or blood components; Bodily fluids such as amniotic fluid, peritoneal fluid, or interstitial fluid; Cell, or a combination thereof. The sample may include a compound that is not naturally mixed with biological materials such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Biological samples may include, for example, urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, airway fluid, intestinal fluid, genitourinary fluid, Intracranial body fluids, ascites, cystic tumor body fluids, saline fluids, or a combination thereof. The sample may be one comprising a circulating tumor cell (CTC).

The TMED2, CD43, or a combination thereof may be present on the surface of a microvessel, for example, a microbeptide derived from a living body. The TMED2, CD43, or a combination thereof may be specifically present on the surface of the microvesicles isolated from an individual having liver cancer. The TMED2, CD43, or a combination thereof may be present at a higher level in a sample isolated from a liver cancer subject than a microvessel isolated from a control sample. The control group may be a normal individual, a disease other than liver cancer, for example, an individual having cirrhosis, or a combination thereof. Therefore, according to the composition, liver cancer can be distinguished from normal individuals or liver cirrhosis individuals.

A substance that specifically binds to TMED2, CD43, or a combination thereof may be a substance that specifically or specifically binds to TMED2, CD43, or a combination thereof, either naturally or non-naturally. The substance that binds transiently may be a substance that specifically binds to TMED2, CD43, or a combination thereof in vivo, for example, a protein. The substance that specifically binds to TMED2, CD43, or a combination thereof may be an antibody, or an antigen-binding fragment thereof, a ligand, a substrate, an inhibitor, an agonist, an antagonist, a cofactor, or a combination thereof. The antibody may be a monoclonal antibody or a polyclonal antibody. The antigen-binding fragment of the antibody may be a single-domain antibody, Fab, Fab ', scFv or a combination thereof.

A substance that specifically binds to TMED2, CD43, or a combination thereof may be one with a detectable label attached thereto. The detectable label may be an optical label, an electrical label, a radioactive label, an enzyme label, or a combination thereof. The optical label may be a substance that generates fluorescence or phosphorescence. The fluorescent material may be, for example, fluorescein, rhodamine, cyanine, metal porphyrin complex, Cy-5 and Cy-3. Examples of fluororesin dyes include 6-carboxyl fluorosine (6-FAM) 1,2, 4 ', 1,4, -tetrahydrochloride (TET) 2 and 2', 4 ' 5 ', 7', 1,4-hexachlorofluorescein (HEX) 3, 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxydodamine (JOE) Chloro-5'-fluoro-7 ', 8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein 5 and 2'-chloro-7'- -Carboxyfluorescein 6 may be included.

The material that specifically binds to TMED2, CD43, or a combination thereof may be immobilized on a glass or solid support. The solid support may be nano or microparticles. The solid support may be magnetic or non-magnetic particles. The solid support may be a bead, a sphere, a polygon, a plate, or a combination thereof. The solid support may have the form of an array in which the material is immobilized in a particular region.

The method also includes measuring the level of the substance in the microvesicles in the sample. The measurement may be to measure the presence or amount of the substance, with the substance specifically binding to TMED2, CD43, or a combination thereof bound to the microvessel. In this case, the substance specifically binding to the TMED2, CD43, or a combination thereof may be a detectable label attached thereto, and the level of the substance can be measured by measuring a signal from the label. The measurement may also be to separate the complex from the mixture in which a substance specifically binding to TMED2, CD43, or a combination thereof is bound to the microbeptide, and then to determine the presence or amount of the substance.

A substance that specifically binds to TMED2, CD43, or a combination thereof may be one with a detectable label attached thereto. The detectable label may be an optical label, an electrical label, a radioactive label, an enzyme label, or a combination thereof. The optical label may be a substance that generates fluorescence or phosphorescence. The fluorescent material may be, for example, fluorescein, rhodamine, cyanine, metal porphyrin complex, Cy-5 and Cy-3. Examples of fluororesin dyes include 6-carboxyl fluorosine (6-FAM) 1,2, 4 ', 1,4, -tetrahydrochloride (TET) 2 and 2', 4 ' 5 ', 7', 1,4-hexachlorofluorescein (HEX) 3, 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxydodamine (JOE) Chloro-5'-fluoro-7 ', 8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein 5 and 2'-chloro-7'- -Carboxyfluorescein 6 may be included.

The level of the substance may be determined by directly separating the substance and measuring the amount thereof, or indirectly measuring the amount thereof without separating the substance. The measurement may be made by detecting a signal from a detectable label attached to the material. Separation of the material may be performed by centrifugation, precipitation, salting out, dialysis, filtration, chromatography, electrophoresis, or a combination thereof. The chromatography may include environmental chromatography, size exclusion chromatography, ion exchange chromatography, or a combination thereof. The measurement may be performed by ELISA, Western blotting, electrophoresis, mass spectrometry, spectroscopy, or a combination thereof.

The method may further comprise, when the level of the substance is higher than that of the control, determining that the subject is suffering from liver cancer. The control group may be a normal individual, a disease other than liver cancer, for example, an individual having cirrhosis, or a combination thereof. Therefore, according to the above method, liver cancer can be distinguished from normal individuals or liver cirrhosis individuals.

The method may further comprise, after the step of contacting, separating the microvesicles from the contact mixture. The separation of the microvesicles may be to specifically separate the microvesicles bound to the material that specifically binds to TMED2, CD43, or a combination thereof. In this case, the separation may be separation using a substance that specifically binds to the substance, for example, an antibody. A substance that specifically binds to the substance, for example, an antibody, may be a solid support such as a magnetic particle or a plate fixed to the plate. The separation may comprise contacting the composite with the material by contacting the material with a material that specifically binds to the material immobilized on the solid support. The microbicule can be separated from the conjugate.

The separation of the microvesicles can be carried out by centrifuging the sample, filtering the sample, incubating the material capable of binding specifically to the microbead or the lipid bilayer of the microbead with the microbead Step, or a combination thereof. The incubation can be performed in vitro. Separation of the microbicule can be carried out by, for example, separation using a solid support or centrifugal force, density gradient method, ultracentrifugation, filtration, dialysis, immunoaffinity chromatography using an antibody, free flow electrophoresis, . ≪ / RTI > The substance that can specifically bind to the microbicule may be a surface protein, a lipid, or a substance capable of binding to the sugar. Wherein the surface protein is selected from the group consisting of CD63, CD83, CD9, integrin-beta 1 (ITGB1), EpCAM, caveolin, FasL, HLA-DRA, CD36, CD63, CD81, MUC1, ERBB4, GPER, ERBB2, MLANA, AMHR2, Lt; / RTI > The substance capable of specifically binding to the microbeptide may be selected from the group consisting of a substance having binding affinity to a protein, a substrate of an enzyme, a coenzyme, a substance which specifically binds to a regulator, a receptor, a lectin, a sugar, a glycoprotein, Binding protein, a protein comprising a pleckstrin homology (PH), a cholesterol-binding protein, or a combination thereof. The antigen-binding fragment comprises an antigen binding site, for example a single-domain antibody, Fab, Fab 'or scFv. The interstitial material in the lipid bilayer of the microvessel may be a material comprising a lipophilic moiety, an amphiphilic moiety, an amphoteric ion moiety, or a combination thereof. The lipophilic moiety may be, for example, a fatty acid, a sterol, or a glyceride. The amphiphilic moiety may be, for example, a phospholipid or a sphingolipid. The amphoteric ion moiety may be, for example, sulfobetaine, carboxybetaine, or phosphorylcholine. A substance capable of specifically binding to the microvesicle or a substance capable of interfering with the lipid bilayer of the microbequilla may be a solid support. The solid support may be spherical, polyhedral, plate, linear, or a combination thereof. The solid support may be polystyrene, polypropylene, magnetic particles or a combination thereof.

The method may further comprise separating the microvessel from the sample separated from the subject prior to the contacting step. In this case, the separation may be such that a substance that specifically binds to TMED2, CD43, or a combination thereof is not used. By doing so, it is possible to measure the level of microvessels present on the surface of a substance that specifically binds to TMED2, CD43, or a combination thereof, in the entire microbequillax. The separation of the microbicules is as described above.

The method may further comprise measuring the level of the tumor marker in the isolated microvessel. The tumor marker may be a liver cancer marker. The term "tumor marker" may be a compound or moiety that is specifically found in tumor cells or tissues. The tumor marker may be a protein or a nucleic acid. The nucleic acid marker may be a miRNA. The tumor marker may be present in the microvessel. The level of the tumor marker may be measured by directly measuring the amount of the marker, or indirectly measuring the amount of the marker without separating the marker. The measurement may be made by detecting a signal from a detectable label attached to the marker. Separation of the markers may be performed by centrifugation, precipitation, salting out, dialysis, filtration, chromatography, electrophoresis, or a combination thereof. The chromatography may include environmental chromatography, size exclusion chromatography, ion exchange chromatography, or a combination thereof. The measurement may be performed by ELISA, Western blotting, electrophoresis, mass spectrometry, spectroscopy, or a combination thereof. If the marker is a nucleic acid, it may be to amplify the nucleic acid and measure the amplification product. The amplification may include an isothermal amplification method, or an amplification method involving thermal cycling such as PCR. The nucleic acid marker can be measured in real time simultaneously with amplification, using a probe containing a FRET pair. The amplification may be real-time PCR.

The method may further comprise measuring the levels of mir-210, mir-346, or a combination thereof in the isolated microvesicles. The measurement may be to directly isolate and measure the amount of mir-210, mir-346, or a combination thereof, or to amplify and measure the amount of mir-210, mir-346, or a combination thereof. The amplification may be carried out using primers and / or probes which bind to mir-210, mir-346, or a combination thereof, or which bind a nucleotide sequence complementary to mir-210, a nucleotide sequence complementary to mir-346, As shown in FIG. The nucleotide sequence may be a natural, synthetic, or semi-synthetic material. The nucleotide sequence may be a nucleic acid, for example, DNA, RNA, DNA-RNA hybrid, PNA, or a combination thereof.

mir-210 represents a mature mir-210 microRNA having the nucleotide sequence of SEQ ID NO: 5. mir-346 represents a mature mir-346 microRNA having the nucleotide sequence of SEQ ID NO: 6.

The nucleotide sequence may be one with a detectable label attached thereto. The detectable label is as described above. The nucleotide sequence may be immobilized on a glass or solid support. The solid support is as described above.

The method may further comprise determining that the subject is suffering from liver cancer when the level of TMED2, CD43, mir-210, mir-346, or a combination thereof in the sample relative to the control is high. The control group may be a normal individual, a disease other than liver cancer, for example, an individual having cirrhosis, or a combination thereof. Therefore, according to the above method, liver cancer can be distinguished from normal individuals or liver cirrhosis individuals.

Another aspect relates to a method of preparing a microbeptide comprising contacting a sample separated from an individual with a substance that specifically binds TMED2, CD43, or a combination thereof to bind the microbeptide and the substance; And measuring the level of the substance in the microbicule in the sample. The present invention also provides a method for obtaining information necessary for diagnosis of liver cancer in an individual.

"Contacting a sample separated from an individual with a material specifically binding to TMED2, CD43, or a combination thereof to bind the material with the microvessel"; And "the step of measuring the level of the substance in the microbicule in the sample" are as described above.

According to one aspect of the composition for diagnosing liver cancer of an individual, it can be used for efficiently diagnosing liver cancer of an individual.

According to another method for diagnosing liver cancer of an individual, liver cancer of an individual can be efficiently diagnosed.

According to another method of obtaining information necessary for diagnosis of liver cancer of an individual, information necessary for diagnosis of liver cancer of an individual can be efficiently obtained.

Figure 1 shows the ROC curve for determining cirrhosis or liver cancer for a sample based on the measured protein band intensity values for the isolated microvesicles using anti-TMED2 coated beads.
Figure 2 shows the ROC curve for determining cirrhosis or liver cancer for a sample based on protein band intensity values measured for isolated microvesicles using anti-CD43 coated beads.
Figure 3 shows the ROC curve for determining cirrhosis or liver cancer for a sample based on protein band intensity values measured for anti-TMED2 coated beads and anti-CD43 coated beads, .
FIG. 4 is a graph showing the results of determining the cirrhosis, or liver cancer, for samples based on the measured values of mir-210 and mir-346 for microbicides isolated using anti-TMED2 and anti-CD43 coated beads The ROC curve for the

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Liver cancer-specific protein Marker  Selection

In this example, liver cancer-specific markers present in microvesicles in blood derived from patients with cirrhosis and patients with liver cancer were identified.

Plasma was isolated from 13 patients with liver cirrhosis and 13 patients with hepatocellular carcinoma by centrifugation at 1300 g for 10 minutes at 4 ° C in BD Vacutainer® Plus plastic blood tubes. The separated plasma was stored at -80 ° C and dissolved in the solution. After centrifugation at 3000g for 5 minutes at 4 ° C, the supernatant was used as a sample except for the precipitate.

300ul of the sample was mixed with 30ul of a bead coated with anti-CD9 antibody (R & D Systems), anti-TMED2 antibody (Santa Cruz Biotechnology), and anti-CD43 antibody (R & D System) The mixture was mixed in a test tube (Axygen), incubated at room temperature for 4 hours using a Grant Bio rotator, and the beads were separated using magnetism. Next, the beads were mixed with 300 ul of PBS and incubated at room temperature for 3 hours to wash. The bead was coated with protein G (Sigma) through a crosslinking reaction using NHS / EDC to Dynabead M-270 Amine (Life technologies, catalog No. 143080, diameter 2.8 μm) Were each reacted and coated with dimethylpimelimidate. The resulting beads contain 2x10 ⁶ beads in 1 l PBS.

After removal of PBS, 30 μl of NuPAGE LDS sample buffer (Life Technologies), which is a lysis buffer containing pH 8.0 and containing lithium dodecyl sulfate, was added to the tube and heat-treated in a heating block at 95 ° C. for 10 minutes, The vesicles were crushed. Western blotting was performed after electrophoresis on the lysate. Detection in Western blotting was carried out by using rabbit anti-integrin-beta 1 as the primary antibody and HRP-conjugated anti-rabbit antibody as the secondary antibody, Respectively. Integrin-beta 1 is a marker commonly found in microvessels.

Based on the measured value of the integrin-beta 1 protein band intensity, the anti-TMED2 antibody or anti-CD12 antibody band intensity value was measured for the integrin-beta 1 protein band intensity value measured on the separated microbeptide using the beads coated with the anti- -CD43 antibody-coated beads were used to calibrate the measured integrin-beta 1 protein band intensity values for the isolated microbeads. Since CD9 is a marker generally present in microvessels, the integrin-beta 1 protein band intensity value measured on the microvesicles isolated using anti-CD9 antibody coated beads is the amount of total microvessels in the sample or Lt; / RTI > in the microvessel.

ROC (receiver operator characteristic) curve analysis was performed using the measured integrin-beta 1 protein band intensity values or a combination thereof for the separated microvesicles using anti-TMED2 and anti-CD43 coated beads.

Table 1 shows the results of cirrhosis or liver cancer determination for samples based on the measured intrinsin-beta 1 protein band intensity values for the separated microvesicles using anti-TMED2 coated beads. In this case, the area under the curve (AUC) is 0.693, the sensitivity is 0.692, and the specificity is 0.692. Figure 1 shows the ROC curve for determining cirrhosis or liver cancer for a sample based on the measured Integrin-beta 1 protein band intensity values for the isolated microvesicles using anti-TMED2 coated beads.

 Actual cirrhosis Actual liver cancer  Predicted cirrhosis 9 4  Predicted liver cancer 4 9

Table 2 shows the cirrhosis or hepatocarcinoma determination results for samples based on the measured intronic-beta 1 protein band intensity values for the microbicides isolated using anti-CD43 coated beads. In this case, the area under the curve (AUC) is 0.538, the sensitivity is 0.308, and the specificity is 0.769. Figure 2 shows the ROC curve for determining cirrhosis or liver cancer for a sample based on the measured Integrin- beta 1 protein band intensity values for the isolated microvesicles using anti-CD43 coated beads.

 Actual cirrhosis Actual liver cancer  Predicted cirrhosis 10 9  Predicted liver cancer 3 4

The integrin-beta1 protein band intensity values measured for the isolated microvesicles using the anti-TMED2-coated beads and the integrin-beta1 protein intensity values measured for the microvesicles isolated using anti-CD43 coated beads Table 3 shows the results of cirrhosis or liver cancer determination based on the protein band intensity value. In this case, the area under the curve (AUC) is 0.692, the sensitivity is 0.692, and the specificity is 0.692. FIG. 3 is a graph showing the effect of the anti-TMED2-coated beads and anti-CD43 coated beads for determining cirrhosis or liver cancer in a sample based on measured Integrin- ROC curve.

 Actual cirrhosis Actual liver cancer  Predicted cirrhosis 9 4  Predicted liver cancer 4 9

From the above results, it was possible to distinguish between liver cirrhosis and liver cancer samples using TMED2 and CD43 as markers.

Example  2: liver cancer specific protein and miRNA Marker  Selection

In this example, liver cancer-specific proteins and miRNA markers present in microvesicles in blood derived from patients with cirrhosis and those with liver cancer were identified.

Plasma was isolated from 13 patients with liver cirrhosis and 13 patients with hepatocellular carcinoma by centrifugation at 1300 g for 10 minutes at 4 ° C in BD Vacutainer® Plus plastic blood tubes. The separated plasma was stored at -80 ° C and dissolved in the solution. After centrifugation at 3000g for 5 minutes at 4 ° C, the supernatant was used as a sample except for the precipitate.

300ul of the sample was mixed with 30ul of a bead coated with anti-CD9 antibody (R & D Systems), anti-TMED2 antibody (Santa Cruz Biotechnology), and anti-CD43 antibody (R & D System) The mixture was mixed in a test tube (Axygen), incubated at room temperature for 4 hours using a Grant Bio rotator, and the beads were separated using magnetism. Next, the beads were mixed with 300 ul of PBS and incubated at room temperature for 3 hours to wash. The bead was coated with protein G (Sigma) through a cross-linking reaction using Dynabead M-270 Amine (Life Technologies, catalog No. 143080, diameter 2.8 μm) using NHS / EDC, Antibodies were each reacted and coated with dimethylpimelidate. The resulting beads contain 2x10 ⁶ beads in 1 l PBS.

After removal of PBS, miRNA was extracted from miRNeasy kit according to manufacturer's instructions and cDNA was synthesized using Universal cDNA synthesis kit (Exiqon). The SYBR Green master mix kit (Exiqon) was then subjected to real time PCR in accordance with the manufacturer's instructions. At this time, the primer provided by the manufacturer was used.

Based on the absorbance value measured at 483-533 nm as measured in RT-PCR, the absorbance value measured for the isolated microbeads using anti-TMED2 coated beads was calculated as the value for mir-210 and the anti- ROC curve analysis was performed by combining the values for mir-346 as the absorbance values measured for the separated microbeads using CD43 coated beads.

The measured absorbance values for the separated microvesicles using the anti-TMED2 coated beads were: the value for mir-210 and the absorbance measured for the microvesicles isolated using anti-CD43 coated beads As a value, the results of cirrhosis or liver cancer determination on the sample based on the value for mir-346 are shown in Table 4. In this case, the area under the curve (AUC) is 0.85, the sensitivity is 0.85, and the specificity is 0.85. FIG. 4 is a graph showing the results of determining the cirrhosis, or liver cancer, for samples based on the measured values of mir-210 and mir-346 for microbicides isolated using anti-TMED2 and anti-CD43 coated beads The ROC curve for the

 Actual cirrhosis Actual liver cancer  Predicted cirrhosis 11 2  Predicted liver cancer 2 11

From the above results, it was possible to distinguish between liver cirrhosis and liver cancer samples using TMED2, CD43, mir-210 and mir-346 as markers.

<110> Samsung Electronics Co., Ltd. <120> A compositon for diagnosing a liver cancer in a subject, method          for diagnosing a liver cancer in a subject and method for          obtaining information for a diagnosis of a liver cancer in a subject <130> PN102613 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 201 <212> PRT <213> Homo sapiens <400> 1 Met Val Thr Leu Ala Leu Ala Leu Val Leu Leu Ala Leu Leu Ala   1 5 10 15 Thr Val Ser Gly Tyr Phe Val Ser Ile Asp Ala His Ala Glu Glu Cys              20 25 30 Phe Phe Glu Arg Val Thr Ser Gly Thr Lys Met Gly Leu Ile Phe Glu          35 40 45 Val Ala Glu Gly Gly Phe Leu Asp Ile Asp Val Glu Ile Thr Gly Pro      50 55 60 Asp Asn Lys Gly Ile Tyr Lys Gly Asp Arg Glu Ser Ser Gly Lys Tyr  65 70 75 80 Thr Phe Ala Ala His Met Asp Gly Thr Tyr Lys Phe Cys Phe Ser Asn                  85 90 95 Arg Met Ser Thr Met Thr Pro Lys Ile Val Met Phe Thr Ile Asp Ile             100 105 110 Gly Glu Ala Pro Lys Gly Gln Asp Met Glu Thr Glu Ala His Gln Asn         115 120 125 Lys Leu Glu Glu Met Ile Asn Glu Leu Ala Val Ala Met Thr Ala Val     130 135 140 Lys His Glu Gln Glu Tyr Met Glu Val Arg Glu Arg Ile His Arg Ala 145 150 155 160 Ile Asn Asp Asn Thr Asn Ser Arg Val Val Leu Trp Ser Phe Phe Glu                 165 170 175 Ala Leu Val Leu Val Ala Met Thr Leu Gly Gln Ile Tyr Tyr Leu Lys             180 185 190 Arg Phe Phe Glu Val Arg Arg Val Val         195 200 <210> 2 <211> 2126 <212> DNA <213> Homo sapiens <400> 2 gcggagctta ggcggcggtg gctgagaagg cagcggggcg gcggcggcgg cggcggcggc 60 ggctgtggag gccgcagtcc gggtcctggc ttcggcctca gccccaccat ggtgacgctt 120 gctgaactgc tggtgcttct ggccgctctc ctggccacgg tctcgggcta tttcgttagc 180 atcgacgccc atgctgaaga gtgcttcttt gagcgggtca cctcgggcac caagatgggc 240 ctcatcttcg aggtggcgga gggcggcttc ctggacatcg acgtggagat tacaggacca 300 gataacaaag gaatttacaa aggagacaga gaatccagtg ggaaatacac atttgctgct 360 cacatggatg gaacatacaa attttgtttt agtaaccgga tgtccaccat gactccaaaa 420 atagtgatgt tcaccattga tattggggag gctccaaaag gacaagatat ggaaacagaa 480 gctcaccaga acaagctaga agaaatgatc aatgagctag cagtggcgat gacagctgta 540 aagcacgaac aggaatacat ggaagtccgg gagagaatac acagagccat caacgacaac 600 acaaacagca gagtggtcct ttggtccttc tttgaagctc ttgttctagt tgccatgaca 660 ttgggacaga tctactacct gaagagattt tttgaagtcc ggagagttgt ttaaaaagcc 720 tcttcctgat gatcccaact cagaattcac tgtttaccaa acaccttggt cataataatg 780 tcattagttt ctccattttt attttctgaa ctgtacattc acaacttatg tttctttgag 840 attaatagat attgggggaa aaacgccttt ttaggaaaat tatagtgaaa atttgacagt 900 tgattggcat aatttcttgt ttgaatgctg cctccattat ataggtcctt ccaggaactc 960 aaacactgta agtgaaatat gggagtatag tttttattat ttcttctttt ccttttgttt 1020 tcataatata atgcagtttg ttcaggaaat cagcacaaag cctgatagta ctttactaaa 1080 atgactgcat tctttggatt ccttcagtct atggttcaag tcactaaaga ttcatttttg 1140 ttgagtcctt atgagaaaca gcagtatgaa tcttgacggt ttctgcccgt cctaatggca 1200 gagctctctg acttgggtgt atgctgccag gctgggtact ttcatacttt gttttcttgt 1260 tttgctttaa aactacgact cagcatacat tttcccacat acatttttac attgtacctt 1320 aggactcagt catctccact taaattgatg acacaagcag ctaataacca tttctgggtt 1380 tctgcctaac cccctaattg tctgttaaag ccaattctct gggtgtccca gtgagtggtg 1440 gctttttttc tttccacatt ggcacattca cttctcccac tcttggcatg taagaaataa 1500 gcatttacat aattggaaaa atctggattt ctgatgccaa agggttaaag cttcttggat 1560 ttcatttcat tgatatacag ccactatttt atttttgatc agtggccttt gggccactgt 1620 tcagggtact gaccatcagt gtcagcatta gggttttggt ttttgtttct tttgggtctt 1680 tcttttttgg cacatgtgaa tcttgttttg tgtaaaatga aattactttc tcttgttctc 1740 tgatgatggg tttaaaatta aaagagcatc cggttttggt atggggatga tccaggatta 1800 tgttgtgact gatacatatt agttacttgt gctttttttt ttttttttgg atctttgcaa 1860 gggcaaaact acaagtaacg agttttatat aattaattta aatttgttac aggttttcat 1920 gtcaggata aaccatactt ccaccttggg tgagaacact tgcaacagtt tattaatgag 1980 gtgactttca ccttaggaca actgttgcat gccaagtttt ttgtgtgtgt gaaacacttc 2040 aaaactgatt taaaagatgt aaatttaaaa ttggttgtat ctaatatgcc ccaggttcgg 2100 taaataaaca attcttttta aaaaca 2126 <210> 3 <211> 400 <212> PRT <213> Homo Sapiens <400> 3 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp   1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro              20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser          35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala      50 55 60 Leu Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser  65 70 75 80 Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln                  85 90 95 Glu Val Ser Ile Lys Met Ser Ser Val Pro Gln Glu Thr Pro His Ala             100 105 110 Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His         115 120 125 Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser     130 135 140 Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Seru Glu Thr 145 150 155 160 Ser Arg Gly Thr Ser Gly Pro Pro Leu Thr Met Ala Thr Val Ser Leu                 165 170 175 Glu Thr Ser Lys Gly Thr Ser Gly Pro Pro Val Thr Met Ala Thr Asp             180 185 190 Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Pro Val Thr Met Thr         195 200 205 Thr Gly Ser Leu Glu Ser Ser Gly     210 215 220 Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala 225 230 235 240 Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met Leu                 245 250 255 Pro Val Ala Val Leu Val Ala Leu             260 265 270 Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala Leu         275 280 285 Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp Ala     290 295 300 Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val Gly 305 310 315 320 Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly Ser                 325 330 335 Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser Arg             340 345 350 Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro Ser         355 360 365 Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala Val     370 375 380 Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala Ala Pro 385 390 395 400 <210> 4 <211> 6944 <212> DNA <213> Homo sapiens <400> 4 agttctcagg ctcacattcc caccacccac ctctgagccc agccctccct agcatcacca 60 cttccatccc attcctcagc caagagccag gaatcctgat tccagatccc acgcttccct 120 gcctccctca ggtcccagct cttgctcctg cctgtttgcc tggaaatggc cacgcttctc 180 cttctccttg gggtgctggt ggtaagccca gacgctctgg ggagcacaac agcagtgcag 240 acacccacct ccggagagcc tttggtctct actagcgagc ccctgagctc aaagatgtac 300 accacttcaa taacaagtga ccctaaggcc gacagcactg gggaccagac ctcagcccta 360 cctccctcaa cttccatcaa tgagggatcc cctctttgga cttccattgg tgccagcact 420 ggttcccctt tacctgagcc aacaacctac caggaagttt ccatcaagat gtcatcagtg 480 ccccaggaaa cccctcatgc aaccagtcat cctgctgttc ccataacagc aaactctcta 540 ggtcccaca ccgtgacagg tggaaccata acaacgaact ctccagaaac ctccagtagg 600 accagtggag cccctgttac cacggcagct agctctctgg agacctccag aggcacctct 660 ggaccccctc ttaccatggc aactgtctct ctggagactt ccaaaggcac ctctggaccc 720 cctgttacca tggcaactga ctctctggag acctccactg ggaccactgg accccctgtt 780 accatgacaa ctggctctct ggagccctcc agcggggcca gtggacccca ggtctctagc 840 gt; cggaacccag atgagaactc acgaggcatg ctgccagtgg ctgtgcttgt ggccctgctg 960 gcggtcatag tcctcgtggc tctgctcctg ctgtggcgcc ggcggcagaa gcggcggact 1020 ggggccctcg tgctgagcag aggcggcaag cgtaacgggg tggtggacgc ctgggctggg 1080 ccagcccagg tccctgagga gggggccgtg acagtgaccg tgggagggtc cgggggcgac 1140 aagggctctg ggttccccga tggggagggg tctagccgtc ggcccacgct caccactttc 1200 tttggcagac ggaagtctcg ccagggctcc ctggcgatgg aggagctgaa gtctgggtca 1260 ggccccagcc tcaaagggga ggaggagcca ctggtggcca gtgaggatgg ggctgtggac 1320 gccccagctc ctgatgagcc cgaaggggga gacggggctg ccccttaagt gtcggtgaat 1380 agtgaggctg gaggccggaa tctcagccag cctccagcac cttccctctc accatcccac 1440 tgccccctcg ctcccatgtt tccacccggc accctgatcc tcacccgaat ctcctttttt 1500 tttttctttt gagacagagt ttcgctttgt cgcccaggct ggagtgcaat gcacgatctc 1560 agttcactgc aacctctgcc tcctaagttc aggcgattct cctgcctcag cttcccgagt 1620 aactgagatt acaggcaccc accaccatgc ccagctgctt ttttgtattt ttggtagaga 1680 tggggtttca ccatgttggc taggctggtc tcaaactcct gacctcaggt gatctacctg 1740 cctcagcctc ccaaagtgct gagattacag acatgagcct ccgcgccttg cctcctcacc 1800 cacctcttca ctctgaatcc tcatgaggct tctcagccct ggatttcctg ctgccatcct 1860 ccccagcac ccacaactag cgcctgggca gggcagggct ggcacctctc aacgtctgtg 1920 gactgaatga ataaaccctc ctcatccacc cctatttatc tccatcacca tttccccctc 1980 tttcttgttc ctggaaacgg ctgctgagtc tccatcggcc aaacttatct gccctgtgat 2040 ttctttgaca attctccttt tcccccagaa cccaccctgg gttgaccaga gtctgggaag 2100 aaggacaaga gaacccggca aactccctcc taggattaac tttgtaaagc acccttgccc 2160 tgtagctgca agggctgtgg aacctgggca gcccgcaacc acctttagct ctgggccccc 2220 caggccagcc tggagcatgg ctgggtgggg ccaccagccc atgctctcag gcgggcctgt 2280 gatctttccc agggcacatg gactgtaggc tggccctggc ccacaccacc acactctccc 2340 cagccatgga cagaggcagc cagaggcctc acggtttctc ctccgagttt ctggctgggt 2400 gtagttctca gaaaccccag tgcctgcgtg tgtccactcg tgggtgtggt ttgtgtgcaa 2460 gagctgagga tttggcgatg cttgggaggg gtagttgtgg gtacagacgg tgtgggggtg 2520 ggaagtggtg cagagactga agagggtcaa cctgggcatg ggggacacag ggactgctga 2580 gaacgtgcgt gtcatctttg ctctgatggg gtggacatag cagaaaatct aactctgtct 2640 gtagccccat acagaatgcc agggtgagca cagtggctgg tgcctttaat cccagcactt 2700 tggaaagttg aggcaggagg atcgcttgag cccaggagtt cgagtctgaa gtgagctgtg 2760 attgcaccac tgcacttcag cctgggcaac agagtgagcc cctgtctcaa aaaagaaaag 2820 aaaaagaaag ccaggcttca tggaaagatc gtatgtgtga cccaaatatg agttcttcag 2880 ctcagccatg gtaatccctt ccttgaagtc tccatttctg cagtacacat gcatgtgcgc 2940 tctctctctc tctctctctc tcacacacac acacacacac acacacacac gcgcgcgcgc 3000 gcgcgctctc ctgcgaacag aggcaggggg agaggggttt gccctggtct cggggactgg 3060 tctggctggc gcttccccac tgcacgtttc caggtttagt ttgtctgtgt ctcctcttcc 3120 atcccagggg ctgagcccct tccatcctcc aagaggaacc agtgagagtg agtgaaggag 3180 gggcctggag ccagggactt cccctgtggg gcctgggtgg agaggggaga actcaatggt 3240 gctgcctttg agaccagccc aggctacagc ccaggagcac acatgggcca gggcagttgg 3300 tatttcccga ggacaaagag gaaattttca aagaggaagt tgttgagtta gagcttgcgg 3360 tggctgagag cagacaggtt gacctgcaaa aaaagacagg ggaggcatgt gagtgtgaca 3420 gccctgctct gtggcctggg caggagatgg gggaaagggt caggtggggg atgggctcgt 3480 gcagtgggag aggagacgga gggagggagc gggaaggggc ttgcttagtg ggtgggaaga 3540 gctgagctcg gatggaacca gcttctacca gccaggctgg gcacccactg ggctgcatct 3600 ggtggccttt tctgattgct atttggactc actgcagctg cagaatgaca gaggccatgt 3660 ccaaaatccc ttagagacac tgttgtctta gagttgttaa aataagagcc cccatatcag 3720 gtttagaaaa tactgtcacc gaacgaacgt cgctgtcctc agctccacct ccctttcctt 3780 tgacagatat ggttgttttc taagccagga ctggttttag tcaggtcctg ggcgaatcct 3840 gaaaaaaaga ggtagtacgg gtaaggaagg cacccaacag ggctttcaca atccagaaaa 3900 tatcaaaata taagtgttaa aagagaggca caggccgggt gcggtggctc acgcctgtaa 3960 tctcagcact ttgggaggcc aaggtgggca gatcatgagg tcaggagttt gagaccagcc 4020 tggccaatat gatgaaaccc cgtttctact aaaaatacaa aagttagcca ggcatggtgg 4080 tgtgctcctg taatcccagc tacttaggag gctgaggcca gagaattgct tgaaccctgg 4140 agtcagaggt tgcagtgagc cgggatcatg ccactgtact ccaggctggg tgacaaagtg 4200 aactio tgaatgcacc aaggaaaatg gtgcattcat aactctcagg tgaagcctac caagccatgc 4320 gtgtgtgcac atatgtgtgt acgtgtgcat gtgcgtgcgt gcatgtgcgt gcgtgcatgt 4380 gcctgtgtgt gtatgtgtgc acatgtgtgt gcgcatgtgt gtgtgtgcgc gcatgtgtgt 4440 gtgcatgcat gttctcccat gcatgtgtac tgtggcaagg gagactttga ggaagagatt 4500 ccagtggctg agcagaaggg ctcgcattgc cctggcgaaa ggttggaagg cttcacctga 4560 gagtgtgtcg tggcctttgt catatccact gcttgattcc tttctttaaa aattattttt 4620 attgttttct acatatgaga accaccacac ctggctaatt tttgtatttt ttgtagagat 4680 ggggtttcac catgttgtcc cggctggtct caaactcccg ggcacaagag atccacctgc 4740 ctcagcctcc caaaatgctg ggactatagg catgagccac tgcacccagc cactgcttca 4800 ttcctggtgg ctgctgtgcc tggcatgttg cagatcctcc atgaatatgc atttgaatga 4860 atgaatgaat gaatgaatga atgaatggag atgacgcctc agagattctt tcttttgaga 4920 tgaggtctca ttctgtcacc cagactagag ggcagtggtg caatcacagc tcaccacagc 4980 ctcaacctcc tgggcctccc aagtagctgc gatcacaggt gtgcaccaac atgcccagct 5040 aatttttttt tttaattttt aatttgtaca gacagggtct tgctgtgttg cccaggctgg 5100 tctcaaactc ctgggctcaa gtggtcctcc cacctaagct tccccaaata ctgggattat 5160 aggtgtgagc cactgtgccc aggcttgcct cagatatttg aaggctggga aggattttgc 5220 aaagctggga aaaggaaaag gcattcccag cagaggggat agcaggtgga aatacataat 5280 taaaaaaaaa aaacgtggag cagatccagc gcagtggctc atgcctgtaa tcccagcact 5340 ttgggaggca gagcagggag gattgcttga gtctaggagt tcaagaccag cctgggtaac 5400 atagaaagac cctgtctcta caaaaacaca aaaaattagc caggcgtggt ggtgcatgcc 5460 tgtagtacca gctacttgag aagctgaggc aggaggactg cttgaagcca ggagtttgag 5520 accagcctgg gcaacatagt gagaccccgt gtctacaaaa agtaaacatt tatatatata 5580 ttttttaaag tggagcagtt caatatagag tcttttttga acaaacgtga aatagatgtc 5640 tttttttttt ttttgagatg gagttttcac tcttgttacc caggctggag tgcaatggcg 5700 tgatcttggc tcaccagaac ctccgcctcc tgggttcaaa caattctcct gcctcagcct 5760 cccaggtagc tgggattaca ggcatgcacc accaaacccg gataattttg tatttttagt 5820 agagatgggg tttcaccatg ttggtcaagc tggtcttgaa ctcccgacct ctgctgatcc 5880 gtatgcctcg gcctcccaaa gtgctgggat tacatgcgtg agccaccgtg cccgacaata 5940 gatgtctttt aattttctgg aggaaaaagc aaagcaaaag aagcagtgga tattttaaga 6000 ctaaaaagga aaacaaaaaa aggagataga gcaggccaga cgtggtggct caacgtctgt 6060 aatcccagca ctttgggagg ccgaggcagg tggatcacct gaggtcagga gttcaagacc 6120 agcctgacca acatggtgaa accctgtttc aaaatacaaa aaattagctg ggcgtggtgg 6180 cgggcacctg taatcccagc tacttgggag gctgaggcag gagaatccct tgaacccagg 6240 aggtggaggt tgcagtgagc cgagatcacg ccattgcact ccagcctggg cgacaagtaa 6300 aaaactccat ctcaaaaaaa aaaaaggaga tagagcaagg aacagtaaga aaatagttgg 6360 gtgcagtggc tatgcggtgg cactatagga ggctgaggcg ggcagatcac ctgaggtcag 6420 gagttggaga ccagcctggg caacatagac ccctatctct acaaaaaatt tgaaatatga 6480 gt; gggaagattg cttgagccca ggagtttgag gctatagtga gctgtgatca tgccactgca 6600 ctccagcctg ggcaatagtg tgagactctg tctcaaaaga aagaacatgg ccaggcgtgg 6660 tggctcacac ctgtaatccc agcactttgg gaggccgagg cagtcagatc atgaggccag 6720 tttgaaacca gcctggccaa catggtgaaa ccctgtctct actaaaaata caaaaattag 6780 ccaggcgtgg tggcatgccc ccgtaatccc agctacttgg gggtctgagg cagaagaatt 6840 gcttgaaacc gggaggcaga ggttgcagtg agccgagatc gtgtcattgc actctagtct 6900 gggcgacaga gcaagactcc gtcttggaaa aaaatttaaa aaaa 6944 <210> 5 <211> 22 <212> RNA <213> Homo sapiens <400> 5 cugugcgugu gacagcggcu ga 22 <210> 6 <211> 23 <212> RNA <213> Homo sapiens <400> 6 ugucugcccg caugccugcc tip 23

Claims (17)

Wherein the composition comprises a substance that specifically binds to TMED2, CD43, or a combination thereof. The composition for diagnosing liver cancer according to claim 1, wherein the substance is a substance that specifically binds to TMED2, CD43, or a combination thereof present on the surface of the microvessel separated from the living body. The composition of claim 1, further comprising a reagent for use in measuring the expression of mir-210, mir-346, or a combination thereof. 4. The composition of claim 3, wherein the reagent binds to mir-210, mir-346, or a combination thereof, or binds to a nucleotide sequence complementary to mir-210, a nucleotide sequence complementary to mir-346, . The composition of claim 1, wherein the substance is an antibody, or antigen-binding fragment thereof, a ligand, a substrate, an inhibitor, an agonist, an antagonist, cofactor, or a combination thereof. The composition of claim 1, wherein the substance is a detectable label. Contacting the sample separated from the subject with a substance that specifically binds to TMED2, CD43, or a combination thereof to bind the microbeptide and the substance; And
And measuring the level of the substance in the microvesicles in the sample. 8. The method of claim 7, further comprising determining that the subject is suffering from liver cancer when the level of the substance relative to the control is high. 9. The method according to claim 8, wherein said control group is measured under the same conditions except that the subject is normal, cirrhosis, or a combination thereof. 8. The method of claim 7, further comprising, after said contacting, separating said microvacles from the contact product. 11. The method of claim 10, wherein said separating comprises centrifugation, separation using magnetism, separation comprising combining to a solid support, or a combination thereof. 12. The method of claim 11, wherein the substance that specifically binds to TMED2, CD43, or a combination thereof is a detectable label. 8. The method of claim 7, further comprising measuring the level of the tumor marker in the microvessel. 14. The method of claim 13, further comprising measuring the levels of mir-210, mir-346, or a combination thereof in the isolated microvesicles. 15. The method of claim 14, further comprising determining that the subject has liver cancer if the level of TMED2, CD43, mir-210, mir-346, or a combination thereof is higher than the control. 15. The method of claim 14, wherein the reagent is attached to a detectable label. Contacting the sample separated from the subject with a substance that specifically binds to TMED2, CD43, or a combination thereof to bind the microbeptide and the substance; And
And measuring the level of the substance in the microbicule in the sample.

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